1. Field of the Invention
This invention relates to compounds that inhibit leukocyte adhesion and, in particular, leukocyte adhesion mediated by alpha 4 integrins. The compounds of this invention are characterized as conjugates containing one or more polyethylene glycol substituents covalently attached thereto. Such conjugates demonstrate enhanced serum half-life and other advantageous pharmacokinetic properties as compared to compounds lacking polyethylene glycol substituents.
2. State of the Art
The physical interaction of inflammatory leukocytes with each other and other cells of the body plays an important role in regulating immune and inflammatory responses [Springer, T. A. Nature, 346, 425, (1990); Springer, T. A. Cell 76, 301, (1994)]. Many of these interactions are mediated by specific cell surface molecules collectively referred to as cell adhesion molecules. These adhesion molecules have been sub-divided into different groups on the basis of their structure. One family of adhesion molecules which is believed to play a important role in regulating immune and inflammatory responses is the integrin family. This family of cell surface glycoproteins has a typical non-covalently linked heterodimer structure.
The particular integrin subgroup of interest herein involves the alpha 4 (α4) chain, which can pair with two different beta chains beta1 (β1) and beta7 (β7) [Sonnenberg, A. ibid]. The α4β1 pairing occurs on many circulating leukocytes (for example lymphocytes, monocytes and eosinophils) although it is absent or only present at low levels on circulating neutrophils. VLA-4 (Very Late Antigen-4, also referred to as α4β1 integrin and as CD49d/CD29), first identified by Hemler and Takada1 is a member of the β1 integrin family of cell surface receptors. VLA-4 consists of an α4 chain and β1 chain. There are at least nine β1 integrins, all sharing the same β1 chain and each having a distinct a chain. These nine receptors all bind a different complement of the various cell matrix molecules, such as fibronectin, laminin, and collagen. VLA-4, for example, binds to fibronectin. VLA-4 also binds non-matrix molecules that are expressed by endothelial and other cells.
VLA-4 (α4β1 integrin) binds to an adhesion molecule called Vascular Cell Adhesion Molecule-1 (or VCAM-1) which is frequently up-regulated on endothelial cells at sites of inflammation [Osborne, L. Cell, 62, 3 (1990)]. VCAM-1 is a non-matrix molecule which is an expressed receptor that is believed to be responsible for trafficking leukocytes into the central nervous system (CNS). α4β1 has also been shown to bind to at least three sites in the matrix molecule fibronectin [Humphries, M. J. et al. Ciba Foundation Symposium, 189, 177, (1995)]. Distinct epitopes of VLA-4 are responsible for the fibronectin and VCAM-1 binding activities and each has been demonstrated to be independently inhibited.2 Based on data obtained with monoclonal antibodies in animal models it is believed that the interaction between α4β1 and ligands on other cells and the extracellular matrix plays an important role in leukocyte migration and activation [Yednock, T. A. et al, Nature, 356, 63, (1992).
The integrin generated by the pairing of α4 and β7 has been termed LPAM-1 [Holzmann, B and Weissman, I. EMBO J. 8, 1735, (1989)] and like α4β1, can bind to VCAM-1 and fibronectin. In addition, .alpha.4.beta.7 binds to an adhesion molecule believed to be involved in the homing of leukocytes to mucosal tissue termed MAdCAM-1 [Berlin, C. et al. Cell, 74, 185, (1993)]. The interaction between α4β7 and MAdCAM-1 may also be important at sites of inflammation outside of mucosal tissue [Yang, X-D. et al. PNAS, 91, 12604 (1994)].
Intercellular adhesion mediated by VLA-4 and other cell surface receptors is associated with a number of inflammatory responses. At the site of an injury or other inflammatory stimuli, activated vascular endothelial cells express molecules that are adhesive for leukocytes. The mechanics of leukocyte adhesion to endothelial cells involve, in part, the recognition and binding of cell surface receptors on leukocytes to the corresponding cell surface molecules on endothelial cells. Once bound, the leukocytes migrate across the blood vessel wall to enter the injured site and release chemical mediators to combat infection. For reviews of adhesion receptors of the immune system, see, for example, Springer3 and Osborn4.
Inflammatory brain disorders, such as multiple sclerosis (MS), meningitis, encephalitis, and a disease model called experimental autoimmune encephalomyelitis (EAE), are examples of central nervous system disorders in which the endothelium/leukocyte adhesion mechanism results in destruction to otherwise healthy brain tissue. Large numbers of leukocytes migrate across the blood brain barrier (BBB) in subjects with these inflammatory diseases. The leukocytes release toxic mediators that cause extensive cell damage and death resulting in impaired nerve conduction and paralysis. Similar occurrences in encephalitis and meningitis indicate that these diseases can be treated with suitable cell adhesion inhibitors.
In other organ systems, tissue damage also occurs via an adhesion mechanism resulting in migration or activation of leukocytes. For example, inflammatory bowel disease15 (including ulcerative colitis and Crohn's disease), are at least partially caused by leukocyte trafficking across the intestinal endothelium via an α4β7 interaction with MadCAM and possibly α4β1 interaction with VCAM-1 expressed in this tissue as well. Asthma6-8, rheumatoid arthritis18-21 and tissue transplant rejection22 are all thought to have components based in interaction of α4β1 with VCAM-1 and/or fibronectin, probably both. it has been shown that the initial insult following myocardial (heart tissue) ischemia can be further complicated by leukocyte entry to the injured tissue causing still further injury (Vedder et al.5). Other inflammatory or medical conditions mediated by an adhesion molecule mechanism include, by way of example, Alzheimer's disease, atherosclerosis9-10, AIDS dementia11, diabetes12-14 (including acute juvenile onset diabetes, tumor metastasis23-28, stroke, and other cerebral traumas, nephritis, retinitis, atopic dermatitis, psoriasis, and acute leukocyte-mediated lung injury such as that which occurs in adult respiratory distress syndrome.
One group of VLA-4 antagonists showing promise as anti-inflammatory agents is the class of sulfonylated-Pro-Phe compounds as set forth in, for example, U.S. Pat. No. 6,489,300.31 These compounds are very potent antagonists of VLA-4/VCAM-1 binding.
Owing to extensive first pass liver metabolism, these compounds are poorly orally available. Because many of the disease conditions treatable by these compounds are chronic conditions, a prolonged serum half-life for the administered compound would increase the usefulness of these kinds of compounds in treating disease in mammals.
The half-life of a drug is a measure of the time that it takes for the amount of drug in the body to decrease by one half, through normal metabolic and elimination pathways. VLA-4 inhibitors, including those disclosed in U.S. Pat. No. 6,489,300, suffer from short half-lives of around 10 to 20 minutes, even when intravenously administered in a pharmaceutical formulation. In order for the patient to retain an effective amount of the drug in their system for a reasonable period of time, either very large quantities of the drug must be administered and/or the drug must be administered many times in a day.
VLA-4 inhibitors with such short half-lives are not commercially viable therapeutic candidates. Therefore, there is a need for VLA-4 inhibitors with significantly enhanced serum half-lives; preferably in the range of hours to days.